ATP is now widely recognized as an extracellular ligand for membrane bound P2-purinergic receptors. Since ATP is coreleased with norepinephrine (NE) from sympathetic nerve terminals in the heart, it may modulate the effects of NE on cardiac cells. We have previously shown that extracellular ATP induces expression of immediate-early genes (IEGs) and inhibits hypertrophic growth of neonatal rat cardiac myocytes. Since cardiac fibroblasts (CAFB) constitute > 70% of the cell population in the mammalian heart, and since Northern blot analysis showed the expression of P2Y purinergic receptor mRNA in CAFB, it was hypothesized that ATP might also exert effects on CAFB via P2-purinergic receptors. To determine whether CAFB respond to P2- purinergic agonists, c-fos expression was studied in cultured neonatal rat CAFB (passage 3). In response to micromolar quantities of ATP, levels of c-fos mRNA were elevated 8-10 fold at 30 min. Relative potencies for c-fos induction in CAFB were UTP = ATP > ATPrS > ADP >> adenosine, consistent with P2-purinergic receptor involvement. ATP increased Cai to peak levels in 10-30 sec and maintained an elevated Cai of lesser magnitude for up to 10 min. BAPTA-AM completely inhibited the ATP-stimulated induction of c-fos. Downregulation or inhibition of protein kinase C with phorbol ester and staurosporine, respectively, partially inhibited the ATP-mediated increase in c-fos mRNA. Western blot analysis demonstrated tyrosine phosphorylation of mitogen-activated protein kinase by 10 min treatment of either NE or ATP. These data show that ATP stimulates c-fos expression via P2-purinergic receptors, and that P2-purinergic receptor stimulation activates multiple second messenger pathways in CAFB. To assess the impact of ATP on CAFB growth, synthesis of DNA and protein was estimated. While NE increased incorporation of labelled precursors into cellular DNA and protein 2-3 fold, ATP inhibited both basal and NE-stimulated incorporation of both precursors. Although UTP was equipotent for induction of c-fos, it did not inhibit incorporation of label into either DNA or protein. These data demonstrate that CAFB respond to P2-purinergic agonists and suggest that distinct P2-purinergic receptors subtypes mediate induction of c-fos and inhibition of DNA and protein accumulation.